Antistasin is a cysteine-rich serine proteinase inhibitor originally isolated from the salivary glands of the Mexican leech Haementeria officinalis (Tuszynski et al., J. Biol. Chem. (1987), 262, 9718-9723). Considerable interest has been focused on this polypeptide of 119 amino acids as it is a highly selective, tight binding inhibitor of the blood coagulation factor Xa and thus a potent anticoagulant in vitro and in vivo (Viasuk et al., Thromb. Haemostasis (1991), 65, 257-262). In addition, antistasin seems to have marked antimetastatic properties (Tuszynski et al., J. Biol. Chem. (1987), 262, 9718-9723).
Amino acid sequence analysis revealed that antistasin contains two homologous domains; no sequence similarity was found to other known proteins suggesting that antistasin is the prototype of a new family of serine proteinase inhibitors (Nutt et al., J. Biol. Chem. (1988), 263, 10162-10167). Meanwhile, only one protein related to antistasin has been identified: ghilanten, isolated from the giant Amazonian leech Haementeria ghilianii is nearly identical to antistasin with respect to amino acid sequence (&gt;90% identity) and functional properties. Recently, a cDNA sequence containing a highly conserved 6-fold repeat with homology to the C-terminal halves of the antistasin-domains has been identified in Hydra (Holstein et al., FEBS Lett., (1992), 309, 288-292); however it, is unclear whether the putative protein encoded by this cDNA sequence has any activity as proteinase inhibitor or antimetastatic agent.
In the present invention novel members of the family of antistasin-type inhibitors are presented. These polypeptides, named hirustasin (Hirudo antistasin), have been isolated from the medical leech Hirudo medicinalis, and their amino acid sequence and characteristics as proteinase inhibitors have been determined. As illustrated in further detail below hirustasin and variants thereof obtained by recombinant DNA technology or peptide synthesis have therapeutic potential in disorders related to the action of serine proteinases such as trypsin, chymotrypsin, tissue kallikrein and cathepsin G, and as antimetastatic agents.
Although the putative reactive site residues of hirustasin and antistasin are nearly identical (FIG. 1), hirustasin neither affects the catalytic activity of isolated factor Xa nor the blood coagulation cascade in vitro.